Previously described methods allow amplification of PCR-amplified cDNA but do not satisfactorily allow quantification (Noonan et al, Proc. Natl. Acad. Sci. 87:7160-7164,1990). Another recently described method (Gilliland et al. Proc. Natl. Acad. Sci. 8 7:2725-2729, 1990) is an improvement in this regard, but still does not include a housekeeping gene internal standard and leaves intact the possibility of error due to variation in aliquoting of the RNA or cDNA sample from one tube to another.
The yield of product from PCR increases exponentially for an indefinite number of cycles. At some point and for uncertain reasons, the reaction becomes limited and PCR product increases at an unknown rate. Consequently, the yield of amplified product has been reported to vary by as much as 6-fold between identical samples run simultaneously. (Gilliland, G., et al., Proc. Natl. Acad. Sci. 87: 2725-2729, 1990). Therefore, after a certain number of PCR cycles, the initial concentrations of target DNA cannot be accurately determined by extrapolation. In an attempt to make PCR quantitative, various investigators have analyzed samples amplified for a number of cycles known to provide exponential amplification (Horikoshi, T., et al., Cancer Res. 52:108-116 (1992); Noonan, K. E., et al., Proc. Natl. Acad. Sci. 87:7160-7164 (1990); Murphy, L. D., et al., Biochemistry 29:10351-10356 (1990); Carre, P. C., et al., J. Clin. Invest. 88:1802-1810 (1991); Chelly, J., et al., Eur. J. Biochem 187:691-698 (1990); Abbs, S., et al., J. Med. Genet. 29:191-196 (1992); Feldman, A. M. et al., Circulation 83:1866-1872 (1991). In general, this is early in the PCR when the PCR product is measurable by use of radiolabeled probes and autoradiography but not by spectrophotometry or densitometry of ethidium bromide stained gels. The use of radioactivity is inconvenient, expensive, and presents safety concerns. Also, the exponential phase must be defined for each set of experimental conditions, requiring additional cost in time and materials.
PCR in the presence of single base mutated competitive templates has been used as an alternative method to make PCR quantitative, Gilliland, supra; Becker-Andre, et al., Nucleic Acids Res. 17:9437-9446 (1989). A known amount of competitive template is co-amplified with an unknown amount of target sequence. The competitor is the same sequence (except for single base mutation) as the target, uses the same primers for amplification as the target cDNA, and amplifies with the same efficiency as the target cDNA. Therefore, the starting ratio of target/standard is preserved throughout the entire amplification process, even after the exponential phase is complete.
Competitive PCR is discussed in general in Siebert, P. D., et al., Nature 359:557-558 (1992); Siebert, P. D., et al., BioTechniques 14:244-249 (1993), and Clontech Brochure, 1993, Reverse Transcriptase-PCR (RT-PCR). All references disclosed herein are expressly incorporated by reference.
However, competitive PCR alone does not adequately control for variation in starting amounts of template. Degradation of samples and pipetting errors can lead to variation. This problem has been circumvented for Northern analysis by probing the same blot for both target gene and a "housekeeping" gene not expected to vary among tissue samples or in response to stimuli. The "housekeeping" gene acts as a denominator in determining the relative expression of a target gene. This constitutive control is just as important when attempting to quantitate or quantify gene expression using PCR. Methods have been described in various of the references cited above, whereby the target gene and the "housekeeping" gene are PCR-amplified in separate tubes. Under these conditions, intertube variation in amplification conditions and pipetting errors remain. Non-competitive multiplex PCR, where the target and "housekeeping" gene are amplified in the same tube, has also been described in Noonan, supra. This method is inconvenient because it requires the generation of standard curves to determine the exponential range of amplification for each target sequence. It also requires the use of radioactive probes.